| Research Abstract |
The methyl-accepting chemotaxis proteins (MCPs) from phylogenetically diverse bacteria have been shown to possess the highly conserved domain (HCD) which is likely to be important for intracellular chemotactic signalling. Based on the conserved 44-amino acid sequence, computer analysis of the P. aeruginosa PAO1 genome sequence predicted that PAO1 possesses 26 open reading frames (ORFs) which likely encode proteins containing HCD. Twenty six ORFs were individually amplified by PCR and cloned into pGEM-Teasy. Individual genes were then disrupted by inserting a kan cassette into the wild-type genes in the PAO1 genome, and the phenotype of kanamycin resistant mutants and PAO1 were analyzed. Chemotaxis assays revealed that pctA, pctB, and pctC were involved in chemotctic responses to ammo acids. The ctpH was required for exhibiting phopshate taxis at high concentrations of inorganic phosphate (P_i), while the gene product of ctpL served as the major chemoreceptor for P_1 at low concentration. The mcp96-3 disruptant failed to respond to O_2, suggesting that MCP96-3 is involved in the detection of C_2. We found that PAO1 was attracted by 2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenoxyacetic acid, and phenoxyacetic acid and repelled by volatile chlorinated aliphatic compounds including trichloroethylene, tetrachloroethylene, trichloroethane, and chloroform. All of the disruptants exhibited normal chemotaxis toward these compounds. These results suggest that each of these compounds is detected by more than two MCPs. PAO1 did not grow on LB broth in the presence of 10% (v/v) toluene. The pilJ mutant showed the sparse growth on LB containing toluene, however, this disruptant emulsified toluene. The pilJ mutant was likely to produce significant amount of biosurfactant, rhamnolipid in the culture condition. The supernatant of the pilJ mutant showed five times more elastase activity detected with PAO1. These results suggest the pilJ gene is involved in quorum sensing.
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